SARAH GUTKOWSKI. B.Sc., University of Minnesota, 2011 A THESIS. submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE

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1 WATER-IN-OIL AND OIL-IN-WATER EMULSIONS STABILIZED BY OCTENYLSUCCINIC ANHYDRIDE MODIFIED STARCH AND ADSORPTION OF MODIFIED STARCH AT EMULSIFIED OIL/WATER INTERFACES by SARAH GUTKOWSKI B.Sc., University of Minnesota, 2011 A THESIS submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Grain Science and Industry College of Agriculture KANSAS STATE UNIVERSITY Manhattan, Kansas 2016 Approved by: Major Professor Dr. Yong-Cheng Shi

2 Abstract Emulsions are utilized to help control phase separation and are found in many products ranging from food to pharmaceuticals. Because of the hydrophobic properties of its functional group, octenylsuccinic anhydride (OSA) modified starch is commonly used in oil in water (o/w) emulsions. The first objective of this study was to investigate if OSA modified starch could be used in water in oil (w/o) emulsions. Experiments were designed to determine the effects of concentrations of OS starch, mineral oil and water on the stability of emulsions. High shear homogenizers and a microfluidizer were used to create stable o/w and w/o emulsions. The stability of the emulsions was examined by optical microscopy, gravitational separation, and electrical conductivity. The microfluidized samples always had a longer stability (days), no gravitational separation and did not exceed three microns, compared to the unmicrofluidized (o/w and w/o) samples. Stable (over 100 days of stability) o/w emulsions could be made without a microfluidizer if the emulsion was made of 2, 60, 38% (w/w) oil, water, starch, respectively. Stable o/w emulsions prepared with a microfluidizer were stable for over 100 days. The o/w emulsion prepared by 8, 66, 26% oil, water, and starch, respectively, was stable for over 600 days. The most stable w/o unmicrofluidized sample was made of 52, 22, 26% oil, water, starch, respectively, with a stability of 240 days. For the w/o emulsions from the microfluidizer, the most stable emulsion was made of 52, 34, 14% oil, water, starch, respectively, with a stability of 250 days. The most stable emulsion that could flow (under the 30,000 cp) was 56, 38, 6% oil, water, starch, respectively, with a stability of 150 days. The statistical mixture experiments models successfully predicted the stability for other ratios of oil, water, and starch for o/w and w/o emulsions. The second objective of the study was to determine the concentration of modified OS starch adsorbed to the mineral oil and the water phases for oil-in-water (o/w) emulsions. The percentage of the starch adsorbed at the mineral oil phase was determined and compared when different ratios of starch to oil and water were used. When the ratio of oil:starch was decreased, the emulsion particle size decreased. As the starch content increased, the percent starch adsorbed onto oil based on total oil increased. The adsorption yield and the level of starch in the emulsion did not show a trend. The surface load ranged from 1.6 to 6.98 mg/m 2. The sample with the

3 highest concentration of starch (26 g/ml) had the highest surface load (6.98 mg/m 2 ) and samples with low concentrations of starch (0.84 and 1.68 g/ml) had the second and third highest surface loads (6.82 and 4.70 mg/m 2, respectively). The ratio of oil:starch was increased to determine the emulsifying capacity. A high emulsifying capacity was achieved. Samples with an oil:starch ratio of 3:1 were stable for over 80 days while other samples with oil:starch ratios of 5:1 and 6:1 could be stable for one week.

4 Table of Contents List of Figures... vi List of Tables... vii Acknowledgements... viii Dedication... ix Chapter 1 - Water-in-oil and oil-in-water emulsions stabilized by OSA modified starch... 1 Abstract... 1 Introduction... 2 Objective... 6 Materials and Methods... 7 Experimental Design... 7 Emulsion Preparation... 8 Characterization of Emulsions... 9 Particle Size Distribution of Emulsions... 9 Optical Microscopy Gravitational Separation Emulsions Viscosity Electrical Conductivity Statistical Analysis Results and Discussion Emulsion Stability O/W Emulsions Viscosity of Emulsions Creaming Index Electrical Conductivity Overall Stability W/O Emulsions Overall Stability Overall Stability for o/w & w/o Microfluidized Samples Conclusions iv

5 References Chapter 2 - Adsorption of modified starch at emulsified oil/water interfaces Abstract Introduction Materials & Methods Procedure Results & Discussion Conclusions Tables References Appendix A: Creaming Index and Conductivity Appendix A.1 Creaming Index and Conductivity of o/w emulsions Appendix B: Adsorption extra calculations Appendix B.1 Calculation of total starch concentration using the Megazyme Kit Appendix B.2 Adsorption yield and surface load equations Appendix B.3 Percent starch adsorbed Appendix C: Adsorption method issues and alternative calculations Appendix C.1 Issues of calculating total starch concentration of emulsions Appendix C.2 Adsorption yield and surface load equations using total starch test reference sample v

6 List of Figures Figure 1.1 Substitution reaction for octenylsuccinic anhydride (OSA) modification... 3 Figure 1.2 Emulsions Mixture Triangle... 8 Figure 1.3 Contour plot of stability (days) of o/w emulsions made without microfluidizer Figure 1.4 Contour plot of stability of o/w emulsions made with microfluidizer in days Figure 1.5 Contour plot of stability (days) of w/o emulsions made without microfluidization Figure 1.6 Contour plot of stability (days) of w/o emulsions made with microfluidization Figure 1.7 Emulsions Mixture Triangle Figure 2.1 Depicting the reference (original) and test samples, with and without centrifugation 31 vi

7 List of Tables Table 1.1 Stability of o/w emulsions Table 1.2 Stability and particle size of o/w emulsions Table 1.3 Stability and viscosity of w/o emulsions Table 1.4 Stability and particle size of w/o emulsions Table 2.1 Surface load and adsorption yield Table 2.2 Adsorption of starch in water and oil phases vii

8 Acknowledgements I would like to first thank my advisor, Dr. Yong-Cheng Shi, for his continued support and guidance. I would also like to thank my committee, Dr. Jon Faubion and Dr. Hulya Dogan, for their encouragement and support. I appreciate the assistance from the statistics department including Nicholas Bloedow and Dr. Leigh Murray. I also very much appreciate all those who worked beside me in the Carbohydrate Polymers Group; you all were a wonderful and supporting Carbohydrate Polymers Family during this journey. I would also like to thank the students, staff, and faculty in The Department of Grain Science and Industry. viii

9 Dedication To my family and friends who have supported me throughout this journey. They have taught me that research, like life, is a journey; there are detours, various paths, some paths that you do not expect, dips and hills along the way. ix

10 Chapter 1 - Water-in-oil and oil-in-water emulsions stabilized by OSA modified starch Abstract Emulsions are utilized to help control phase separation and are found in many products ranging from food to pharmaceuticals. Because of the hydrophobic properties of its functional group, octenylsuccinic anhydride (OSA) modified starch is commonly used in oil in water (o/w) emulsions. The first objective of this study was to investigate if OSA modified starch could be used in water in oil (w/o) emulsions. Experiments were designed to determine the effects of concentrations of OS starch, mineral oil and water on the stability of emulsions. High shear homogenizers and a microfluidizer were used to create stable o/w and w/o emulsions. The stability of the emulsions was examined by optical microscopy, gravitational separation, and electrical conductivity. The microfluidized samples always had a longer stability (days), no gravitational separation and did not exceed three microns, compared to the unmicrofluidized (o/w and w/o) samples. Stable (over 100 days of stability) o/w emulsions could be made without a microfluidizer if the emulsion was made of 2, 60, 38% oil, water, starch, respectively. Stable o/w emulsions prepared with a microfluidizer were stable for over 100 days. The o/w emulsion prepared by 8, 66, 26% oil, water, and starch, respectively, was stable for over 600 days. The most stable w/o unmicrofluidized sample was made of 52, 22, 26% oil, water, starch, respectively, with a stability of 240 days. For the w/o emulsions from the microfluidizer, the most stable emulsion was made of 52, 34, 14% oil, water, starch, respectively, with a stability of 250 days. The most stable emulsion that could flow (under the 30,000 cp) was 56, 38, 6% oil, water, starch, respectively, with a stability of 150 days. The statistical mixture experiments models successfully predicted the stability for other ratios of oil, water, and starch for o/w and w/o emulsions. 1

11 Introduction Emulsions are used in food, beverage, industrial, and pharmaceutical applications to bring two substances that are usually not miscible together and typically one ingredient is dispersed in another. The two most common types of emulsions are oil-in-water (o/w) and waterin-oil (w/o) emulsions, with w/o being the most common in food applications (Stauffer, 1999). By nature, oil and water tend to separate after mixing; therefore, there is a great need for emulsifiers to keep them from separating. The classification of an emulsion depends upon which substance is in the dispersed phase or the continuous phase. In a w/o emulsion, water is dispersed in the oil; thus, water is the dispersed phase and oil is the continuous phase. Emulsions are formed by applying mechanical force to a system composed of oil, water, and an emulsifier. Some types of mechanical force include high-pressure homogenizers, high-shear homogenizers, microfluidizers, and sonicators. The emulsion may become unstable due to internal and external factors such as time, temperature, and makeup of the emulsion (McClements, 1999). Two types of instability are physical and chemical instability. Physical instability is more common than chemical instability for emulsions using starch. When molecules are distributed, placed, or organized differently compared to its preferred state, this is an example of physical instability (McClements, 2005). Physical instability includes coalescence, flocculation, sedimentation, creaming, Oswald Ripening, or phase inversion (McClements, 1999, 2005 & 2007). Oswald Ripening tends to happen for flavor emulsions and the emulsions prepared in this study are cloud emulsions, which use non-flavor oils (Buffo & Reineccius, 2001; McClements, 2005). Understanding the interactions of the ingredients is important to determine the kinetic stability of the emulsion, the rate of the process/change that happens (McClements, 2005). The emulsifier attached to the oil droplet prevents it from adhering to another oil droplet (McClements, 2005). According to McClements (2007), creaming is defined as particles of lower density (ie, oil) coming together and floating to the top, packing together to form a creamed layer because they cannot rise anymore. If the dispersed phase particles are of higher density than the continuous phase, the droplets will sink to the bottom through sedimentation (McClements, 1999). Creaming is common for o/w emulsions while sedimentation is common for w/o emulsions. Creaming may have been caused by flocculation or coalescence (McClements, 2005). A particle size analyzer cannot tell if the emulsion has undergone 2

12 flocculation or coalescence. Instead, a microscopic image of the emulsion, before and after aggregation, shows the droplets in either the flocculated or coalesced form. If the particles are observed to have increased in size, the emulsion is undergoing coalescence. If the particles stay the same size, but are aggregated, the emulsion is undergoing flocculation (McClements, 2005). Another way to distinguish between flocculation and coalescence is viscosity of the emulsion. Higher emulsion viscosity is usually due to flocculation, while a lower emulsion viscosity is usually coalescence (McClements, 2005). The occurrence of flocculation, which is considered a quality failure factor in the food industry, can be inhibited by lowering the droplet size, radius, and critical concentration (Chanamai & McClements, 2002). Modified starch may be utilized as an emulsifier by means of its functional groups, which bridge the interface of two immiscible phases. One way to create an amphiphilic starch is to react the starch (hydrophilic) with octenylsuccinic anhydride, or OSA (hydrophobic) (Trubiano, 1986). Figure 1.1 Substitution reaction for octenylsuccinic anhydride (OSA) modification (Bai, 2007; Nilsson & Bergenstahl, 2007) The starch s hydroxyl groups provide the hydrophilic component while the octenylsuccinate group provides the hydrophobic component (Figure 1.1). The amphiphilic nature of the OSA modified starch helps the starch act as an emulsifier that can be used in beverages (Trubiano, 1995). The OSA reacts with hydroxyl groups attached to carbons 2, 3, and 6 on the starch s glucose units and undergo a substitution reaction (Trubiano, 1986; Bai, 2007). FDA guidelines dictate that up to 3% OSA can be added to react with starch for food applications and the product must be labeled Food Starch Modified (Trubiano, 1986). The 3

13 degree of substitution (DS) for 3% OSA modified starch is usually about (Nilsson, Leeman, Wahund, & Bergenstahl, 2006). OSA modified starches have shown to be good stabilizers and emulsifiers by inhibiting and slowing the natural process of instability. The main stabilization mechanism is through steric hindrance where the droplets are protected from aggregation (Chanamai & McClements, 2001; Tesch, Gerhards, & Schubert, 2002). Steric hindrance works to stabilize molecules using nonionic molecules and help inhibit particle aggregation, but cannot directly inhibit sedimentation and creaming (Chanamai & McClements, 2001; Napper, 1976; Tesch, Gerhards, & Schubert, 2002). In addition, OS starch increases the viscosity of the continuous phase which inhibits the movement of molecules and therefore, reduces the interaction of particles and stabilizes the emulsions (McClements, 2005). The OS starch can reach the interface of the system because it has a high molar mass and is easily adsorbed (Nilsson et al., 2006). Based on kinetic factors, polymers with a greater radius have a decreased adsorption time (the time it takes for the particles to adsorb to the surface) when the sample is processed under turbulent flow (Nilsson et al., 2006). With a larger sized molecule, there is a higher substituent density and higher adsorption energy. Therefore, there will be a larger surface load, which is the amount absorbed to an interphase and measured in units of macromolecule per surface area (milligrams per square meter) (Nilsson et al., 2006). This concept is related to overrepresentation of large molecules at the surface and stronger kinetic adsorption factors (Nilsson & Bergenstahl, 2007). Therefore, the larger size of the OS starch molecules, and their larger radius, causes the OSA modified starch to be adsorbed into the interfacial region faster and so can be used as efficient emulsifiers (Nilsson et al., 2006). OS starch can function as a surfactant. When the surfactants are at the interfacial region (area between the hydrophilic and hydrophobic regions, where the emulsifier acts), they lower the interfacial or surface tension (γ). This lower surface tension helps stabilize emulsions (Prochaska, Kedziora, Thanh, & Lewandowicz, 2007). Many emulsifiers are available on the market, including gum arabic, mono- and diglycerides, hydrocolloids, methyl cellulose, and modified starches (McClements, 2007; Saunders, 1968). All of these have benefits and drawbacks. Gum arabic has been used in many applications as an emulsifier; however, gum arabic is not always consistent in its quality (Chanamai & McClements, 2002). Gum arabic has been shown to be adequately replaced by hydrophobically modified starches, such as OS starch, in carbonated beverages (Trubiano,

14 & 1995). The beverages may be stabilized with a lower concentration of OS starch than the concentration of gum arabic for an equivalent purpose with no change in particle size, clouding efficiency, or emulsion stability (Trubiano, 1986). OS starch, compared to gum Arabic, has a higher oil load (National Starch Bulletin) and a high surface load (Nilsson and Bergenstahl, 2006 and 2007). OSA modified starch has been used in the beverage emulsions, flavors, and clouding agents (Prochaska et al., 2007). OSA modified starch has been used for emulsifying and encapsulating flavors and nutrients such as vitamin E (Qiu, Yang, & Shi, 2015). Reiner, Reineccius, & Peppard (2010) compared gum arabic and starch-based emulsifiers for cloud emulsions, also called o/w emulsions, and found that the native and modified gum acacia is more stable than the modified starches when used in orange terpene based beverages. The modified starches and hydrocolloid emulsifiers examined were commercial samples. Emulsion stability is defined by McClements (2005 & 2007) as resisting physiochemical change. There are various ways to measure emulsion stability, depending on the sample. Optical microscopy, gravitational separation, rheology, and electrical conductivity are used to measure the stability of the emulsions (McClements, 2007). Bessoles, Duccini, & Trouve (2011) noted that emulsions are stable if no breakage is noticed after 6 months at room temperature. Emulsions should flow; otherwise, they may be more of a gel and not a flowable emulsion (McClements, 2007). Prochaska et al. (2007) found that the viscosity of the starch solution changes from the ph of the system. Using a Brookfield viscometer, they found increased viscosity (48, 200, 225, 275, and 350 mpas) with increased ph (2.5, 3.5, 4.5, 5.5, and 7 ph respectively) of their starch solutions. The degree of substitution and the pasting viscosity is compromised with less than ideal ph conditions, which is outside of the ph of 5.5 to 7.0 range (Prochaska et al., 2007). OS starch is not stable at high ph (Chung, Lee, Han, & Lim, 2010). Changes in OS starch can influence the rheological properties and the gelling process of OS starch and is affected by the temperature and the time of the processing (Martinez, Partal, Munoz, & Gallegos, 2003). Conductivity can be used to determine if the emulsion is o/w or w/o by determining the electrical potential of the sample (McClements, 2007). The conductivity of a sample can also help determine if the sample is going through phase inversion. Oil in water emulsions have an aqueous continuous phase; therefore, they have a high conductivity. A lipid continuous phase from a w/o emulsion creates a lower conductivity. If the o/w emulsions were to destabilize, this 5

15 would result in a lower conductivity. If the emulsion has a high conductivity (o/w emulsions) and reduces in conductivity over time to the range that s common for w/o emulsions, it may have gone through a phase inversion. Hydrophilic-Lipophilic Balance (HLB) has been calculated and used to predict if the emulsifier is a lipophilic emulsifier (HLB value < 10) or a hydrophilic emulsifier (HLB value > 10) (Wang et al., 2011). HLB is a good predictor, but it alone cannot predict if the emulsifier is good in a w/o or o/w system. OS starch has a HLB hydrophilic emulsifier value of 12.7 (Wang et al., 2011); however, the w/o has not been evaluated so this needs to be checked. OS starch is usually used for o/w emulsions, however there are emulsifiers used for w/o emulsions as well (Fingas & Fieldhouse, 2003; Iyer, Hayes, & Harris, 2001; Yan, Gray, & Masliyah, 2000). Iyer et al. (2001) made w/o emulsions as small as micrometers using methoxypolyethylene glycol, triethylamine, mesyl chloride, p-toluenesulfonic acid, 3- hexadecanone, lysozyme, N-acetylglucosamine, and glycol chitin (degree of polymerication of 2500). Those are all non-starch based w/o emulsions. Yan et al. (2000) used kaolinite clay particles treated with hydrophobic polystyrene latex miceospheres and could make w/o emulsions as small as two microns. However, most of the surfactants they used did not allow them to create stable emulsions. Fingas & Fieldhouse (2003) studied w/o emulsions (called chocolate mousse in the oil industry) stabilized by asphaltene, resin, and oil emulsions for the oil industry where emulsification slows oil spills at sea. They found that stable emulsions have between 60 and 80% water. They did not examine OS starch. Objective OSA modified starch is commonly used in oil in water (o/w) emulsions. The objective of this study was to investigate if OS starch could be used in w/o as well as o/w emulsions. Experiments were designed to determine the effects of the levels of OS starch, oil, and water on o/w and w/o emulsions. High shear homogenizers and a microfluidizer were used and compared. The stability of the emulsions was determined using multiple methods including optical microscopy, viscosity, gravitational separation, and electrical conductivity. 6

16 Materials and Methods OS starch, HI-CAP 100, was obtained from Ingredion (Bridgewater, NJ) and had a DS of 0.02, and 6.0% moisture. Mineral oil (cat. No. BP2629-1) and sodium benzoate (cat. No. S ) were purchased from Fisher Scientific (New Jersey, USA). Distilled water was made in the laboratory. Experimental Design For experiments with three ingredients, the experimental domain is within an equilateral triangle (Fig. 1.2). The vertices represent the pure components, the edges of the triangle represent the two-component blends, and points within the triangle represent the three-component blends. This is called the Mixture Emulsion Design. A completely randomized design (CRD) following the mixtures experiments with a centroid design were used (Figure 1.2). This mixture design was chosen because this research uses more than two ingredients in a mixture where the levels of one factor are dependent of the other factor s levels (Mason, Gunst, & Hess, 2003). The design was used for both un-microfluidized and microfluidized samples. To reduce sample pool size, MiniTab Mixtures Experiment Triangle Statistical Software (Minitab, Inc., Pennsylvania) predicted the stability of all possible emulsions by using only 50 samples (plus replicates and other preliminary samples). The samples start with two and not zero percent so every ingredient will be included equally and due to preliminary research, at least 2 percent starch was needed. There were two areas of compositional interest, o/w and w/o, as shown in Figure 1.2 as bold smaller triangles. 7

17 Figure 1.2 Emulsions Mixture Triangle depicting area of largest triangle. The smaller upper left triangle are w/o emulsions and the smaller lower left triangle are o/w emulsions. The amount of each ingredient are shown as true percentages. These areas were determined by preliminary data, previous methods, and data from Shah, Tsong, Sathe, & Liu (1998) and McClements (2007). The design was formed with ten points each. The augmented simplex centroid design was used for the component mixture sample. The response variable was days of stability. Figure 1.2 shows those parts of the emulsion mixture triangle focused on in this study. The other areas are explained in the discussion section. Emulsion Preparation Water and the OS starch (Figure 1.2, Table 1.1 and Table 1.3) were stirred at 25 C in a 150 ml screw cap glass jar on a magnetic stirring plate for 1.5 h. The samples that were not transparent after 1.5 h of stirring (due to higher concentration of starch) were stirred for an additional hour. Once the starch solution was semi-translucent, sodium benzoate was added at a level of 0.1% of the starch concentration. The mixture was then stirred in a water bath at 60 C for 15 min to ensure complete dissolution. Mineral oil was added slowly while a High Shear Homogenizer (Bamix Biohomogenizer, Switzerland) mixed the sample at high speed (10,000 rpm) for 6 min at 25 C, until there were no oil droplets on the surface. The total weight of each emulsion was 150 g. Due to the high viscosity (around 30,000 cp at a shear rate of 2 s -1 ), the w/o emulsions also were mixed with a Laboratory Bench Top Homogenizer (PRO Scientific Inc., Oxford, CT, USA) for 4 min at 8,000 rpm. For comparison purposes, samples were also made without the lab bench top homogenizer. 8

18 Part of each sample was saved for stability testing via optical microscopy, viscosity, electrical conductivity, and gravitational separation measurements. The remainder of the sample was microfluidized (Microfluidizer M110PII, Microfluidics, Westwood, MA) at 18,000 psi for 5 passes. The chambers used were Auxilary Processing Module (APM = H30Z), 200 microns followed by the Interaction Chamber (G10Z), 87 microns. Cold tap water surrounding the coil of the microfluidizer ensured sample temperature was maintained at 30 C. Care was taken to insure that only the emulsion was collected from the microfluidizer. The microfluidizer originally had water in its piping; therefore, the initial output from the microfluidizer was discarded. This ensured that the collected emulsion represented the undiluted original sample. Due to the high viscosity of the w/o emulsions, some samples (M and Q) were made with only 3 passes, instead of 5 because the emulsions were too thick by the third pass and would have occluded the microfluidizer if they were subjected to a fourth or fifth pass. The same stability tests were conducted on the non-microfluidized (and microfluidized) samples. The samples were stored at 4 C for subsequent stability measurements. Characterization of Emulsions Particle Size Distribution of Emulsions A Laser Scattering Particle Size Analyzer LA-910 (Horiba Ltd., Kyoto, Japan) was used to determine the particle size (volume diameter) of the o/w emulsions at room temperature (~25 C), but was not used for w/o emulsions because the samples would be diluted by water. The pipette was inserted into the middle of the o/w emulsion and when the tip was withdrawn from the sample, the pipette tip was wiped with a Kimwipe. Due to possibility of oil droplets on the surface of the emulsions, only the middle of the emulsion was sampled for this test. This was injected into the reservoir tank using distilled water as the dispersant. Only o/w emulsions can be tested with the particle size analyzer. To ensure a homogenous sample, each sample and dispersing liquid was agitated at 400 rpm. The sample was then circulated and sonicated with ultrasonic vibrations (39 khz). The particle size (assuming all particles were spherical) was determined by the instrument s software equations that were based on the light scattering off the particles. The light sources were a He-Ne laser and tungsten lamp and the software produced a particle size distribution. The particle size was evaluated on fresh and aged emulsions, depending on the results from the gravitational separation stability testing. Therefore, particle size was 9

19 tested on day 0, 7, and if there was a difference in gravitational separation. The samples were run in duplicates. Optical Microscopy Each emulsion was viewed by an optical microscope (Olympus BX51 microscope, Olympus America Inc., Melville, NY). The emulsion sample varied between 0.1 to 2 ml depending on the original starch concentration and opacity of the sample. The sample was also taken by a pipette and placed on a plain microscope slide and covered with a cover slip (Fisher Sci. International Inc.). A 40x objective lens was used for all samples. The SPOT 4.6 Windows software (Diagnostic Instrument Inc., Sterling Heights, MI, USA) captured the images of the emulsions. Calibration markers were added to the images, with the software, to give the particle sizes of the droplets. The samples were run in duplicates. Gravitational Separation The gravitational separation was performed by the method of McClements (2007) using graduated transparent centrifuge tubes. The creaming index was calculated using the serum layer value and the total height of the emulsion. The samples were run in duplicates. A known homogenous concentration of the sample (40 ml) was poured into the 50 ml plastic screwcap tubes and sealed. The tube was inverted once back and forth to ensure a homogenous sample. The samples were kept undisturbed at 4 C to ensure a constant temperature and observations were taken daily. Changes in appearance or if layers appeared were measured and the time noted. The creaming index (CI) was calculated by the following formula: CI = 100 x HS/HE Where HS is the serum layer and HE is the emulsion layer. McClements (1999) and Dickinson (1992) describe the Stokes law for the creaming rate of a particle in a liquid and how it can predict an emulsion s stability. If the original particle size of the oil droplet is 1 micron and has a density ρ of 910 kg/m 3, suspended in water (shear viscosity η1 = 1 mpa s, density ρ = 1000 kg/m 3 ) then the creaming rate will be 17 mm/day. Dickinson (1992) determined that if an emulsion has a creaming rate less than 1 mm/day it can be called an emulsion stable against creaming. 10

20 Emulsions Viscosity The viscosity of emulsion was measured at room temperature (~ 25 C) by DV-II+Pro Brookfield viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) with the #21 spindle. The samples were tested at different rpms in order to have a torque value between 10 and 100%. The viscosity, shear stress and shear rate were recorded. The samples were run in duplicates. Electrical Conductivity The conductivity of a sample was measured by an electrical conductivity meter (Fisher Scientific, accumet, Pittsburgh, PA, USA) based on the method of McClements (2007). The output number was in ms/cm or μs/cm. The samples were run in duplicates. Statistical Analysis Every sample was produced in triplicate. The samples for all of the analysis methods were run in duplicates. The results were analyzed using Microsoft Excel (Redmond, WA) where calculations were conducted for the mean, standard deviations, and other statistical analysis. For experiments with three ingredients, the experimental domain is within an equilateral triangle. The vertices represent the pure components, the edges of the triangle represent the twocomponent blends, and points within the triangle represent the three-component blends. This is called the Mixture Emulsion Design. MiniTab (State College, PA) was used to produce the Mixture Emulsion Design. The results were also analyzed using SAS Program Software MiniTab using the Mixture Emulsion Design results. 11

21 Results and Discussion Emulsion Stability The stability (in days) of the emulsions was determined by gravitational separation, particle size growth/expansion in microns, conductivity change, and creaming index. An emulsion was deemed stable if it was stable for at least 100 days. It is stable if there is no separation of phases. The stability of the other parts of the emulsion triangle that are not completed in the lab was estimated by inputting the results for the selected mixtures (see Methods section). O/W Emulsions Table 1.1 Stability of o/w emulsions, where the amount of water, oil, and starch are in percent, on a 100 g basis. Stable emulsion? w/out with ID oil water starch microfluidizer A No No B No Yes C No Yes D No Yes E No Yes F No Yes G No Yes H No Yes I No Yes J Yes Yes All of the o/w emulsions without microfluidizing were not stable for the 100 days except for sample J (Table 1.1). This may be due to the large amount of emulsifier needed to create o/w at low mechanical energy (without the microfluidizer). All of the o/w emulsions created using the microfluidizer were stable except for sample A (Table 1.1). Sample A may not have enough starch (emulsifier) needed to emulsify the sample. 12

22 Table 1.2 Stability and particle size of o/w emulsions. The amount of water, oil, and starch are in percent, on a 100 g basis. "X" indicates that the sample is not stable. Particle size (microns) Particle size (microns) w/out microfluidizer w/ microfluidizer ID Stable Day 0 Day 100 Stable Day 0 Day 100 in days average average in days average average A ±0.5 a x ±0.5 a x B ±0.5 a x ±0.0 b 1.0±0.0 b c C ±1.0 a x ±0.1 a 2.5±0.1 a D 8 1.0±0.5 a x ±0.0 b 2.0±0.5 a b E ±1.0 a x ±0.0 b 0.2±0.0 c F ±1.0 a x ±0.0 b 0.15±0.0 c G ±1.0 a x ±0.0 b 0.9±0.0 b c H ±1.0 a x ±0.0 b 0.15±0.0 c I ±0.5 a x ±0.0 b 2.5±0.9 a b J ±0.5 a 2.0± ±0.0 b 3.0±0.9 a Means that do not share a letter are significantly different. The emulsions that were not microfluidized had initial particle sizes around 2 microns, whereas the microfluidized samples had particle sizes between 0.15 and 1.5 microns (Table 1.2). The particle size increased over time (0 to 100 days) for all the samples. There was no trend relating particle size to amount of starch for the unmicrofluidized and microfluidized samples. The microfluidized samples with the largest particle size (A and C with particle sizes of 1.5 microns) had the highest amount of oil (38 and 26%, respectively) (Table 1.2). Samples A and C were unstable emulsions because they had the highest oil content. For the o/w emulsions that were not microfluidized, the most stable emulsions were ones with more starch and water (Table 1.2). The sample with 2% oil, 60% water, and 38% starch had an average of 120 days of stability (2.0 +/- 0.5 microns for particle size) (Table 1.1 and 1.2). The main reason is the high viscosity contributed by high percentage of starch. The movement of oil droplets in the system was reduced, thus the chance of droplets getting close is low. This means that this OSA modified starch can be used for o/w emulsions with common laboratory equipment (hand held homogenizer to make a crude emulsion and bench top high shear mixing homogenizer), and does not have to go through microfluidization. For the o/w emulsions that were microfluidized, the most stable emulsions were almost evenly spread through various starch: oil: water combinations conducted in these experiments. The most stable samples were 14% oil, 72% water, and 14% starch, which was stable for over 13

23 365 days (3.0 +/- 0.9 microns for particle size); and the sample with 2% oil, 96% water, and 2% starch, which was also stable for over 365 days (0.15 +/- 0 microns particle size) (Table 1.2). Microfluidized emulsions were more stable than those not undergoing this process. These particle sizes were smaller and stayed smaller for a longer period of time (Table 1.2). This agrees with the results from Taherian, Fustier, & Ramaswamy (2005). Many of the o/w emulsions had particle sizes less than 1 micron. When the emulsion was visibly unstable by the gravitational separation test its particle size was larger (usually by tenfold) than a fresh emulsion (Appendix A). Most of the emulsions had a large increase in particle size at the point of their instability. Some emulsions had an initial particle size greater than 2 microns and some as high as 9 microns after 100 days. Samples D (20, 60, 20; oil, water, starch, respectively) and F (14, 72, 14; oil, water, starch, respectively) had very short shelf lives. Both had a ratio of starch to oil of 1:1. Viscosity of Emulsions The emulsions with higher concentrations of starch and oil (samples A and J), making the emulsion very viscous, had a lower particle size when going through 3 passes instead of 5 passes. Some of the viscous samples (A and J) could not pass through the 87 micron size channel more than 3 passes and instead the emulsion was broken. Therefore, the w/o samples had 3 passes; however these same samples are made with 5 passes as well in order for comparison. The oil in water emulsions did not have a viscosity value due to being not viscous enough for the Brookfield Viscometer (due to the equipment, the value is under 2 cp at 100 rpm, SS and SR are estimated at 2.33 and 93.0, respectively. Creaming Index The creaming index results did not show a clear trend (appendix A). The samples became unstable in three different ways. The w/o emulsions tended to form two layers while the o/w emulsions formed three layers, unless the samples had component ratios close to the phase inversion line. The two layers were composed of cream and serum layers. The three layer system was composed of oil, emulsion, and serum layers. The creaming index was similar for the majority of the samples. It stayed at zero until the emulsion was unstable. The trends of the remaining emulsion samples are in appendix A. Once the samples showed signs of a layer of oil on the surface, (i.e. a creaming index above zero) the samples were declared unstable. This 14

24 determination in conjunction with the other analyses allowed the estimation of stability in days. This value was used in the subsequent statistical models. Electrical Conductivity Conductivity tended to increase over time for all samples except 38, 60, 2; 20, 78, 2; and 2, 96, 2 (percent oil, water, starch, respectively) (Appendix A). The conductivity of these samples was constant. The increase in conductivity may be due to the way these emulsions become unstable. When these emulsions are in their unstable state, as determined by gravitational separation, an oil layer was present on the surface, then an emulsion layer (middle), and finally a serum layer (bottom). When the sample was assessed for conductivity, the probe was submerged in the middle of the sample and the sample stirred slightly. If there were a few oil droplets coalescing and rising to the surface, these may not have been detected due to slight stirring of the sample. Therefore, the sample in contact with the probe had a higher water content. Water (conductivity of 7.18 µs for distilled water and 0.41 µs for deionized water), but not oil (0 µs), can conduct electricity. Therefore, the samples had a higher ms conductivity over time. Overall Stability Figure 1.3 Contour plot of stability (days) of o/w emulsions made without microfluidizer Figure 1.3 is the contour plot of stability of o/w emulsions made without the microfluidizer. The R 2 (adj) was 72%, suggesting that the stability for o/w emulsions without the 15

25 microfluidizer may be reasonably predicted with this model. A higher R 2 indicates a better fit. The Lack of Fit (LOF) was low (Figure 1.3). This was a good indicator that this model accurately estimated stability for the remaining emulsions in the plot. Figure 1.3 shows the stability in days using darker gray and lines representing longer stability. This contour plot of stability shows MiniTab results estimating the days of stability for every point in the mixtures triangle. The most stable emulsion was 2, 60, 38 (percent oil, water, starch, respectively), (the low right hand corner of the triangle, and marked with the darkest gray). Figure 1.3 shows that the more stable emulsions are those with more starch and less oil. Figure 1.4 Contour plot of stability of o/w emulsions made with microfluidizer in days Figure 1.4 shows the contour plot of stability of o/w emulsions made with the microfluidizer. The R 2 (adj) value of 83%, was sufficient to make accurate estimations of stability for the remainder of the emulsions in the triangle. Figure 1.4 shows the stability in days using a darker green and checkered representing longer stability. The most stable emulsion was sample H (2% oil, 96% water, 2% starch). The more stable emulsions are those with more water (Figure 1.4). When comparing Figure 1.4 to Figure 1.3, microfluidized emulsions are more stable. 16

26 W/O Emulsions Table 1.3 Stability and viscosity of w/o emulsions, where the amount of water, oil, and starch are in percent, on a 100 g basis. Note: the emulsion is called stable if it is stable for at least 100 days, without separation. "Thick" indicates that the emulsion was too viscous for the viscometer. "X" indicates that the emulsion was not prepared due to being too thick for the microfluidizer. SR is the shear rate and SS is the shear stress. Viscosity Day 0 Stable emulsion? w/out microfluidizer w/ microfluidizer w/out with SR SS viscosity SR SS viscosity ID oil water starch microfluidizer (s -1 ) (Pa) (cp) (s -1 ) (Pa) (cp) K No No ±2 a 1500±5 c 93 40±1 b 43±2 d L No Yes 20 90±1 d 440±3 d ± ±7 L ± ±9 L 1 331± ±7 L ±5 a 29467±8 a M No X thick thick thick X X X N No Yes ±6 b 1500±4 c thick thick thick O No No 93 30±2 e 31±2 f 20 17±2 c 90±3 c P Yes Yes ±6 d 509±4 d thick thick thick Q Yes X thick thick thick X X X R No No 1 239± ±7 thick thick thick R ± ±7 R 5 427±3 c 9200±9 b S No Yes 20 40±3 e 200±3 e 1 259± ±7 S ± ±8 S 3 406±3 a 14583±5 b T Yes Yes 1 134± ±3 thick thick thick T 3 300± ±8 T 5 439±3 c 9440±4 a Means that do not share a letter are significantly different. For the w/o emulsions created without the microfluidizer, the most stable contained more starch and water (Table 1.3). Thus, OSA modified starch can be used for w/o emulsions made with common laboratory equipment, and does not have to go through microfluidization. Specifically, the area with 52% oil, 22% water, and 26% starch had an average of 240 days of stability. All of the w/o emulsions made without microfluidization were not stable except for samples P, Q, and T, potentially due to the large amount of emulsifier needed to emulsify the sample with low mechanical energy (without the microfluidizer) (Table 1.3). Samples P and M had the same amount of starch, but M was not stable. This is possibly due to the higher amount 17

27 of oil in sample M. All of the w/o emulsions made with the microfluidizer were stable except for samples K, O, and R. This may be due to the sample not having enough starch (emulsifier) to emulsify the sample. Except for sample O, all other samples were viscous and some (samples M and Q) too thick for the Brookfield viscosity measurement. The emulsion viscosity over 50,000 cp could not be determined with the Brookfield. Because the samples were tested at different rpms in order to have a torque value between 10 and 100%, some of the samples were too thick or too thin in order to be measured at 20 SR. Table 1.3 shows the viscosity of the samples around 20 SR. Sample L microfluidized could not get up to a SR of 20 and therefore the table shows the results that were obtainable. Sample O unmicrofluidized only had one viscosity measurement because everything else was out of range. But, sample O microfluidized had results at multiple SR and therefore, the viscosity at 20 SR was calculated. Other samples could not go through the microfluidizer (labeled thick on Table 1.3), most likely because they were too thick and clogged the instrument. Thus if the system has high amount of oil, a lower starch content is needed, to lower viscosity. Table 1.4 Stability and particle size of w/o emulsions, where the amount of water, oil, and starch are in percent, on a 100 g basis. "X" indicates that the sample is not stable. "N/A" indicates that the emulsion was not prepared due to being too thick for the microfluidizer. Particle size (microns) w/out microfluidizer Particle size (microns) w/ microfluidizer Day Day 0 Day 100 Day Day 0 Day 100 ID unstable average average unstable average average K 4 2.5±1.0 a b X ±0.1 a X L ±0.5 a X 100 1±1 a 3±1.5 a M ±0.5 c X N/A N/A N/A N ±0.1 c X ±0.5 a 3±1 a O 10 1±0.5 b c X ±0.5 a X P ±0.1 c 3±1 a ±0.1 a 2±1 a Q ±0.1 c 0.75±0.5 a N/A N/A N/A R 7 0.8±0.1 b c X 15 1±0.5 a X S ±0.5 a X ±0.01 a 3±1.5 a T ±0.01 c 2±1 a ±0.01 a X Means that do not share a letter are significantly different. 18

28 W/o emulsions could be created without the use of a microfluidizer (Table 1.4). The best sample was P (52, 34, 14; percent oil, water, starch, respectively) which was stable for 100 days and had a viscosity of 505 cp (30 rpm, 27.9 s -1 SR, Pa SS). An even more stable sample was sample Q (52, 22, 26; percent oil, water, starch, respectively). It was stable for 240 days, but could not flow (over the max viscosity limit for the Brookfield). OS starch could be used to create stable w/o emulsions, best with a microfluidizer (Table 1.4). However, some of these stable emulsions such as the most stable (250 days of stability) sample P (52, 34, 14% oil, water, starch, respectively) could not flow (14,583 cp, 3 rpm, s- 1 SR, Pa SS) (Tables 1.3 and 1.4). Sample O (52, 46, 2% oil, water, starch, respectively) was stable for 50 days and could flow (Tables 1.3 and 1.4). Sample L (64, 34, 2% oil, water, starch, respectively) was stable for 100 days, but would flow very slowly, 29,467 cp (1.5 rpm, 1.39 s-1 SR, Pa SS). Sample S (56, 38, 6% oil, water, starch, respectively) was stable for 150 days, but was viscous and could only flow only very slowly (Tables 1.3 and 1.4). There was no trend for the conductivity of these samples. Emulsions created without microfluidizing had initial particle sizes between 0.2 and 2.5 microns as measured by microscopy. The particle size increased over time (0 to 100 days) for all the samples. By 100 days, the microfluidized samples had particle sizes between 0.08 and 1 microns. The nonmicrofluidized samples that had higher amounts of starch (samples M, N, P, Q, and T) had initial original particles sizes ( microns), but no such trend was not observed for the microfluidized samples. 19

29 Overall Stability Figure 1.5 Contour plot of stability (days) of w/o emulsions made without microfluidization Figure 1.5 shows the contour plot of stability of w/o emulsions made without microfluidization. The R 2 (adj) value was 96% so this model was accurate to estimate the days of stability for the remainder of the emulsions in the triangle. The Lack of Fit (LOF) was zero. Figure 1.5 shows the stability in days using darker gray and checkered representing longer stability. This mixture contour plot of stability shows the results from MiniTab results estimating the days of stability for every point in the mixtures triangle. The lower right hand corner of the triangle (darkest gray and checkered) represents Sample G, which had 52, 22, 26% oil, water, starch, respectively. The more stable emulsions are those with more starch and less oil. The lightest gray dotted areas are the least favorable combinations of oil, water, and starch if made without using a microfluidization (Figure 1.5). The light gray region was where there was too little starch and more oil, making the sample difficult to stir during the homogenization steps and being too viscous to pass through the microfluidizer, without clogging. 20

30 Figure 1.6 Contour plot of stability (days) of w/o emulsions made with microfluidization Figure 1.6 shows the contour plot of stability of w/o emulsions made with the microfluidizer. There was still a problem with the two lower points samples (2 samples, each having a duplicate), which are omitted from this analysis. This was due to that the samples were not able to go through the microfluidizer. The overall model was significant. The R 2 (adj) value was 91%, which was sufficient for this study, to make accurate estimations of stability for the remainder of the emulsions in the triangle. This means that the regression model fits well. The Lack of Fit (LOF) was high. Figure 1.6 shows the stability in days using a darker green representing a longer stability. This mixture contour plot of stability shows the results from MiniTab results estimating the days of stability for every point in the mixtures triangle. To read the graph, here was an example: the lower left hand corner of the triangle was the darkest green and checkered. This point was representing the 52, 34, 14 (percent oil, water, starch, respectively) sample. The more stable emulsions are those with less oil and a balance between water and starch (Figure 1.6). In Figure 1.6, the white and dotted area shows the least favorable combinations of oil, water, and starch. The lower right region, are combinations with too much starch, making the sample difficult to stir during the homogenization steps and very viscous to pass through the microfluidizer, which causes clogging. Due to clogging for the too thick samples, these are left out of the analysis. 21

31 Overall Stability for o/w & w/o Microfluidized Samples Figure 1.7 Emulsions Mixture Triangle of the amount of water, oil, and starch are in percent, on a 100 g basis. This study focused on microfluidized samples: w/o emulsions (smaller upper left triangle) and o/w emulsions (smaller lower left triangle) where the key indicates that the solid black areas are stable over 100 days stable and the gray solid areas are unstable under 100 days stable. The other areas were not prepared and thus cannot be distinguished as o/w or w/o emulsions. The upper right area (vertical lines) are samples that cannot go through the microfluidizer. The lower right area (dots) are samples where the starch:water ratio is too high. The lower left area (horizontal lines) are samples where results from the contour plots show low stability, and the final middle area (diagonal lines) are samples where it is not clear if the emulsion is o/w or w/o. The area outside of the small o/w and w/o triangles (Figure 1.7) were not the focus of this study. The samples in the upper right hand corner have too much oil which results in a very unstable emulsion while using this modified starch and the sample will be very viscous and not fluid (usually over 30,000 cp at 1.5 rpm, shear stress around 410 Pa and a shear rate around 1 s -1 ; more viscosity results in Table 1.3). The instrument chosen for this experiment had limitations as 22